Project Summary Peripheral artery disease (PAD) is a form of cardiovascular disease that can reduce blood flow in the lower limbs ultimately resulting in the potential for loss of limb. Early clinical trials in patients with PAD resulting in critical limb ischemia have demonstrated the safety of autologous stem cell therapies with modest improvements in reperfusion and limb salvage. While stem cell therapies may therefore represent a realistic alternative to conventional revascularization therapies, a number of challenges remain which limit large-scale clinical trials and widespread use. Outcomes with respect to clinical efficacy have been less than ideal and are largely attributed to the well-documented cell loss following delivery. The overwhelming majority (>90 %) of cells do not survive implantation after 1-2 weeks. We have described that a degradable hydrogel matrix can improve stem cell-mediated muscle function recovery following ischemic injury. Our data suggested that one mechanism for improved recovery was the increased number of cells that were maintained at the site of injury and their beneficial effects on the host response. Central to our understanding is the necessity of being able to track stem cells and the infiltrating inflammatory cells, particularly macrophages. Major challenges to the development of clinically applied stem cell therapy remains the lack of technologies for stem cell tracking, methods for the improvement of cell viability and strategies to understand the stem cell-mediated host response. Therefore, the overall goal of the current proposal is to understand the role of delivered stem cells and the mechanisms of repair in vivo using nanotracer-enhanced, high-resolution combined ultrasound and photoacoustic (US/PA) imaging. A secondary goal is to be able to quantify the role of recruited macrophages in a model system in which we are able to correlate imaging quantification with quantitative measures of muscle function. We propose here to utilize plasmonic gold nanoshells as nanotracers, due to their excellent biocompatibility, as well as tunable, strong optical absorption properties. In contrast with other imaging techniques, combined US/PA imaging can visualize and quantify cell delivery and function over a broad range of timescales, spatial resolutions and imaging depths. High-resolution imaging of tissues is possible as well as visualization in 3D. The combination of nanotracers with US/PA imaging results in a unique approach that will allow us to answer fundamental questions regarding MSC involvement in muscle repair as well as validate a clinically translatable solution for tissue regeneration. We propose to develop this single system in a mouse model of hindlimb ischemia in which we are able to quantify muscle function, thereby allowing correlation with tissue regeneration.